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Background: Understanding the source of newly detected human papillomavirus (HPV) in middle-aged women is important to inform preventive strategies, such as screening and HPV vaccination., Methods: We conducted a prospective cohort study in Baltimore, Maryland. Women aged 35-60 years underwent HPV testing and completed health and sexual behavior questionnaires every 6 months over a 2-year period. New detection/loss of detection rates were calculated and adjusted hazard ratios were used to identify risk factors for new detection., Results: The new and loss of detection analyses included 731 women, and 104 positive for high-risk HPV. The rate of new high-risk HPV detection was 5.0 per 1000 woman-months. Reporting a new sex partner was associated with higher detection rates (adjusted hazard ratio, 8.1; 95% confidence interval, 3.5-18.6), but accounted only for 19.4% of all new detections. Among monogamous and sexually abstinent women, new detection was higher in women reporting ≥5 lifetime sexual partners than in those reporting <5 (adjusted hazard ratio, 2.2; 95% confidence interval, 1.2-4.2)., Conclusion: Although women remain at risk of HPV acquisition from new sex partners as they age, our results suggest that most new detections in middle-aged women reflect recurrence of previously acquired HPV., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)